| Interferons have antiviral, antigrowth, immunomodulatory and antiangiogenic effects. The human Type I interferons, IFN-alpha, IFN-beta, and IFN-o, compete for a single heterodimeric receptor, IFN-o, composed of two subunits IFNAR-1 and IFNAR-2. Human IFNAR-2 can bind a variety of Type I IFNs, but with lower affinity and different specificity than the IFNAR complex. Human IFNAR-1 has low intrinsic binding of human IFNs, but plays an important role in determining the final ligand specificity and affinity of the IFNAR complex. Understanding its interactions with the IFNs is critical to elucidating the differential ligand specificity of IFNAR. Fortuitously, the homologous bovine IFNAR-1 alone is capable of high-affinity binding of human Type I IFN's, enabling a study of the residues of IFNAR-1 involved in ligand binding.;Using the bovine IFNAR-1 we created and analyzed a series of 56 mutations for a loss-of-binding phenotype. Using this method, we have identified functionally important residues that enable the bovine IFNAR-1 to bind human IFN-alpha2. Five aromatic residues in the two central subdomains of bovine IFNAR-1 had very strong effects on ligand binding: mutagenesis of proximal neutral or charged residues had smaller effects. Our identification of several critical aromatic residues comprising the core of the receptor ligand-binding site is consistent with previously identified binding sites found on other cytokine receptors.;These key binding residues were mapped onto a homology model of IFNAR-1 to identify the ligand-binding face of IFNAR-1. Of the five aromatic residues, four were found to be easily accessible to a single ligand the size of interferon. These four residues, W132, F139, Y160, and W253, comprise a core of ligand binding in IFNAR-1, and are conserved across species. This alanine-scanning mutagenesis represents the only direct analysis of IFNAR-1 ligand binding, and provides the first view of a localized ligand binding face of IFNAR-1. |
